The epicardium is a single cell-layer mesothelial sheet that surrounds the heart and harbors a multi-potent progenitor cell population. Amazingly, epicardial-to-mesenchymal transition (EMT) leads to cardiac fibroblast and coronary vessel formation at the precise moment that diffusion fails to fuel the heart. The epicardial fetal gene program is re-awakened in ischemic heart disease and contributes to coronary neoangiogenesis and fibrosis. Surprisingly, none of the currently known regulatory mechanisms explain how EMT occurs in perfect synchrony with physiological demand. Serum response factor (SRF) is a widely expressed transcription factor that controls gene expression programs through interactions with tissue specific or signal responsive co- factors. Embryonic and adult cardiovascular function depends upon interactions between SRF and myocardin, which is specifically expressed in cardiomyocytes and smooth muscle cells, constitutively nuclear, and required for activation of genes encoding contractile proteins. Conversely, myocardin-related transcription factor (MRTF)-A and MRTF-B are broadly expressed, but held dormant in the cytoplasm until physiological signals lead to their nuclear accumulation. MRTF-A and -B promote differentiation of a mesenchymal / myofibroblast cell type in response to a growing list of agonists, including Rho-Rho kinase, TGF-1, and mechanical tension. Our recently published data reveal a critical role for MRTF-A in myofibroblast differentiation and scar formation following myocardial infarction. Our preliminary studies reveal SRF, MRTF-A and MRTF-B are enriched in the embryonic and adult epicardium and are required for EMT. Further, MRTF/SRF activity is induced by hypoxia and promotes a mesenchymal phenotype in cooperation with Wilms tumor 1 (WT1), including the coordinated regulation of guidance cues (Wnt signaling) and cytoskeletal components. Based on our preliminary data and the work of others, we hypothesize that physiological hypoxia in the epicardium promotes the synergistic interaction between MRTFs, SRF, and WT1 that drives EMT, coronary vessel formation and cardiac fibroblast production during development and disease. We will test this hypothesis with three Specific Aims that will define the molecular mechanisms underlying transcriptional regulation in the epicardium. Aim 1 will determine how MRTFs and SRF control epicardial cell function during development using conditional deletion of these factors and lineage tracing experiments in mice. Aim 2 will determine the transcriptional mechanism governing epicardial cell fate and function by defining the expression signature cooperatively regulated by SRF, MRTFs, and WT1 in the epicardium, and identify the physiological cues that modulate this gene regulatory axis. Aim 3 will define the role of MRTF-SRF in epicardial derived cell differentiation and cardiac function following myocardial infarction. These studies will test a paradigm-shifting hypothesis that explains the coordinated regulation of epicardial cell migration and differentiation by physiological cues and reveal novel therapeutic targets for the treatment of ischemic heart disease.